Apoptosis In Multiple Myeloma Cell Lines Research Articles

Arginine Methylation Mediated By PRMT5 Has Significant Biological and Clinical Impact in Multiple Myeloma

Arginine-specific methyltransferases critically regulate cellular homeostasis by dictating the biological outcome of target proteins. Among them, Protein Arginine Methyltransferase 5 (PRMT5) has attracted growing interest due to its role as an enzyme mediating epigenetic regulation of anti-cancer target genes, as well as in methylation of non-histone proteins involved in growth-regulating and survival pathways including p53. However, little is known about its biologic function in multiple myeloma (MM).To first evaluate the clinical significance of PRMT5 in MM pathogenesis, we analyzed RNA-seq data from newly-diagnosed MM patients and identified highly upregulated PRMT5 in 320 patients' CD138+ cells compared to 16 samples of normal bone marrow (BM) plasma cells. Additional analysis of PRMT5 expression in two independent datasets also showed further PRMT5 mRNA upregulation during progression of MM. Immunohistochemical staining also confirmed elevated expression of PRMT5 in BM biopsies from MM patients as compared to healthy individuals and monoclonal gammopathy of undetermined significance (MGUS). Moreover, analysis of the prognostic significance of PRMT5 expression in MM patients enrolled on IFM/DFCI 2009 clinical study showed that high PRMT5 expression was associated with poor prognosis in terms of both event free (p= 0.016) and overall survival (p=0.018). Consistently, we also found upregulated PRMT5 expression at both mRNA and protein levels in MM cell lines (N=11) and patients CD138+ MM cells (N=3) as compared to PBMCs from healthy volunteers, associated with a parallel increase of cellular symmetric arginine di-methylation (SDMA) substrates. Interestingly, genetic depletion of PRMT5 in H929 (p53wt) and KMS11 (p53null) MM cell lines by shRNA decreased SDMA levels, associated with cell growth inhibition in a p53-independent manner. Likewise, pharmacological inhibition of PRMT5 with the small molecule inhibitor EPZ015666 triggered decreased SDMA levels, cell growth, survival, and clonogenicity, as well as induction of caspase-dependent apoptosis in MM cell lines. Moreover, although PRMT5 and SDMA levels were increased in MM cells cultured in the presence of BM stromal cell supernatant, cytotoxic activity of EPZ015666 was maintained. Notably, drug treatment significantly impaired cell proliferation of patient MM cells (n=2) even in the presence of BM mononuclear or stromal cells, without toxicity on normal PBMCs. At the level of gene expression modulation, PRMT5 inhibition was associated with downregulation of NF-kB-dependent transcription, evidenced by both gene set enrichment analysis (GSEA) and Ingenuity Upstream Regulator Analysis. Moreover, analysis of protein levels confirmed reduction of both canonical and non-canonical NF-kB pathways, evidenced by significantly decreased NF-kB DNA binding activity by ELISA. Importantly, Mass Spectrometry analysis identified TRIM21 as a new PRMT5 interactor; and EPZ015666-treated cells showed that PRMT5 methylates TRIM21 evidenced by WB analysis. Since TRIM21 mediates monoubiquitination of IKKbeta, thereby triggering its selective autophagy-mediated degradation, we next analyzed EPZ015666 effects on IKKbeta. Treatment increased both monoubiquitination of IKKbeta and the formation of IKKbeta-TRIM21-pBECLIN1-pULK1 autophagic complexes. Conversely, inhibition of autophagosome formation by 3-methyladenine abrogated the anti-MM activity of EPZ015666 and IKKbeta degradation, indicating that selective autophagic degradation of IKKbeta and inhibition of NF-kB signaling mediates EPZ015666-triggered anti-MM activity. Consistent with this view, confocal microscopy analysis also confirmed co-localization of IKKbeta in the autophagosome after EPZ015666 treatment. Finally, stable silencing of TRIM21 in MM cell lines significantly abrogated the anti-proliferative effect of EPZ015666. Collectively, these data delineate arginine methylation as a new control mechanism of MM cell growth, and demonstrate that inhibiting PRMT5 decreases tumor cell survival via blockade of NF-kB signaling, even in the context of the BM milieu. These data demonstrate the biologic and prognostic significance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome in MM. DisclosuresHideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:Takeda: Consultancy; Celgene Corporation: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Consultancy, Equity Ownership. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Global Genomic Analysis of Newly Diagnosed t(4 ;14) Multiple Myeloma Reveals a Specific Mutational Spectrum and Identifies PKD2 As a Potential Therapeutic Target

▪In Multiple Myeloma (MM), the t(4;14) translocation is associated with a poor outcome. However, beside this translocation, the genetic events which determine the adverse evolution of the disease and the resistance to treatments remain elusive.In this study we performed whole exome or RNA sequencing analysis of samples from 65 newly diagnosed t(4;14) MM. We found that NRAS, KRAS, MAPK and FGFR3 are frequently mutated (12%, 9%, 13.8%, and 20% respectively). Overall, the FGFR3/RAS/BRAF/MAPK genes were mutated in 36 cases (54%). There was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF as expected from the mutually exclusive occurrence of mutations in these genes. In addition to alterations in TP53 and DIS3, we found marked elevated frequency of mutations in PRKD2 (10.7%), ATM/ATR (10.7%) and MYCBP2 (7.6%), reduced frequency in FAM46C (1.5%) and no mutation in TRAF3 and CCND1. Mutations in ATM/ATR were strongly associated with the MB4-2 breakpoint (Bp) (p = 1.62 10-4) and significantly correlated with mutations affecting genes coding for members of the MAPK family. We observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or MB4-3 Bp (p = 1.3 10-2). Of note, PRKD2 mutations are exclusively found in 3 t(4;14) MM cell lines and among the 84 MM sequenced by Bolli et al. (1), none of the non t(4;14) patient were mutated in PRKD2, indicating that this genetic lesion is associated with t(4;14) MM.In the NCI-H929 t(4;14) MM cell line, which is mutated for PRKD2, encoding the PKD2 serine/threonine kinase, we observed elevated levels of phosphorylated PKD2. Furthermore, inhibition of PKD, decreased PKD2 phosphorylation and triggered reduced proliferation and apoptosis of MM cell lines and fresh plasma cells from patients in vitro. These results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients.Altogether, these results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients.Reference1. Bolli, N., Avet-Loiseau, H., Wedge, D.C., Van Loo, P., Alexandrov, L.B., Martincorena, I., Dawson, K.J., Iorio, F., Nik-Zainal, S., Bignell, G.R., et al. (2014). Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun 5, 2997. DisclosuresMunshi:Janssen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties.

Repurposing Leflunomide for Multiple Myeloma Treatment

Background: Despite recent advances in treatment that have improved the prognosis for patients with multiple myeloma (MM), the disease remains incurable. There is a need for MM treatments with new mechanisms of action. Leflunomide, a commercially available oral immunosuppressive agent that has been FDA-approved since 1998 for the treatment of rheumatoid arthritis (RA) was evaluated as a potential MM therapy. The primary mechanism of action is de novo inhibition of pyrimidine synthesis by targeting dihydroorotate dehydrogenase (DHODH), and thus achieving an anti-proliferative effect in B- and T-lymphocytes. A secondary mechanism of action is inhibition of cytokine and growth factor receptor-associated tyrosine kinase activity. Methods and Results: Pre-clinical studies of teriflunomide, the active metabolite of leflunomide, showed that it inhibited cell growth and induced apoptosis in MM cell lines (MM.1S, MM.1R, U266, H929, RPMI-8226) and primary MM patients’ (CD138+) plasma cells at clinically achievable concentrations (50-200 uM) in a time- and dose-dependent manner. We also found that teriflunomide induces cell-cycle arrest in both, glucocorticoid-sensitive (MM.1S) and resistant (MM.1R) MM cell lines at <200 uM. In addition, teriflunomide and dexamethasone synergized in the in vitro growth inhibition of MM cell line MM.1S. To identify MM-associated mRNAs and miRNAs whose expression levels are frequently altered upon teriflunomide exposure, MM cell lines (RPMI-8226, U266, MM.1S, NCI-H929) and CD138-enriched primary plasma cells from two MM patient samples were treated with 200 µM teriflunomide or DMSO control for 24 h before extraction and purification of mRNA and microRNA. mRNA-seq and miRNA-seq analysis from teriflunomide-treated MM samples revealed that similar changes were present between patient samples and cell lines. A total of 382 genes were found to be differentially expressed (225 upregulated, 157 downregulated). Upregulated genes included those that participate in defense response and negative regulation of cell growth. Genes involved in mitosis, rRNA biogenesis/processing, and immune response were generally downregulated. Analysis of microRNA-seq data from these samples revealed five differentially expressed, mostly newly discovered miRNAs that have unknown function.Conclusions: Leflunomide and its analogues demonstrated anti MM effects in vitro as well as synergy with dexamethasone. Based on our promising pre-clinical results we have initiated a single-agent phase I/II clinical trial in patients with relapsed/refractory MM. DisclosuresNo relevant conflicts of interest to declare.

Cannabinoid derivatives exert a potent anti-myeloma activity both in vitro and in vivo.

Although hematopoietic and immune system show high levels of the cannabinoid receptor CB2, the potential effect of cannabinoids on hematologic malignancies has been poorly determined. Here we have investigated their anti-tumor effect in multiple myeloma (MM). We demonstrate that cannabinoids induce a selective apoptosis in MM cell lines and in primary plasma cells of MM patients, while sparing normal cells from healthy donors, including hematopoietic stem cells. This effect was mediated by caspase activation, mainly caspase-2, and was partially prevented by a pan-caspase inhibitor. Their pro-apoptotic effect was correlated with an increased expression of Bax and Bak, a decrease of Bcl-xL and Mcl-1, a biphasic response of Akt/PKB and an increase in the levels of ceramide in MM cells. Inhibition of ceramide synthesis partially prevented apoptosis, indicating that these sphingolipids play a key role in the pro-apoptotic effect of cannabinoids in MM cells. Remarkably, blockage of the CB2 receptor also inhibited cannabinoid-induced apoptosis. Cannabinoid derivative WIN-55 enhanced the anti-myeloma activity of dexamethasone and melphalan overcoming resistance to melphalan in vitro. Finally, administration of cannabinoid WIN-55 to plasmacytoma-bearing mice significantly suppressed tumor growth in vivo. Together, our data suggest that cannabinoids may be considered as potential therapeutic agents in the treatment of MM.

BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth.

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy.

Bone Marrow Stromal Cells Induce Bortezomib Resistance in Multiple Myeloma Cells through Downregulation of miRNA-101-3p Targeting Survivin

INTRODUCTIONIt is not yet fully understood how bone marrow microenvironment components especially bone marrow stromal cells (BMSCs) induce drug resistance in multiple myeloma (MM). This form of drug resistance has been suggested to pave the way for intrinsic (de novo) resistance to therapy in early stages of the disease and contribute to acquired drug resistance in the course of treatment. Hence, deciphering the molecular mechanisms involved in induction of above resistance will help identify potential therapeutic targets in MM combined treatments. Our previous work showed that BMSCs (normal and MM patient-derived) induced resistance to bortezomib (BTZ) compared with MM cells in the absence of stroma. This resistance was associated with modulation of a transcriptome in MM cells, including prominent upregulation of oncogenes c-FOS, BIRC5 (survivin) and CCND1. However; whether these oncogenes mediate BTZ resistance in the context of BMSCs through interaction with miRNAs is not known.METHODSHuman myeloma cell lines, 8226, U266 and MM.1s, were co-cultured with MM patient-derived BMSCs or an immortalized normal human line (HS-5) in the presence of 5nM BTZ for 24 h. MM cell monocultures treated with 5nM BTZ were used as controls. Co-cultures were then applied to magnetic cell separation (EasySep, Stem Cell Technologies) to isolate MM cells for downstream analyses (western blotting and qPCR). Total RNA including miRNAs was isolated from MM cell pellets (QIAGEN miRNeasy kit), cDNAs were synthesized (QIAGEN miScript RT II kit) and applied to miScript miRNA PCR Array (SABioscience, MIHS-114ZA). After normalization of all extracted Ct values to 5 different housekeeping genes, fold changes in miRNA expression were analyzed in co-cultures compared to MM cell monocultures using the 2-ΔΔCt algorithm. Moreover, survivin gene was silenced in MM cells using Ambion® Silencer® Select siRNA and Lipofectamine RNAiMAX transfection reagent. Survivin-silenced cells were then seeded on BMSCs and exposed to BTZ. Percent apoptosis of gated CD138+ MM cells was determined using FACS. For our overexpression and 3'UTR reporter experiments, we transiently transfected MM cells with pre-miR-101-3p, scrambled miRNA or pEZX-3'UTR constructs using Endofectin reagent (all from GeneCopoeia).RESULTSBMSCs upregulated survivin gene / protein (a member of inhibitors of apoptosis family) and modulated an array of miRNAs in MM cells compared to MM cells in the absence of stroma. The more noticeably downregulated miRNAs were hsa-miR-101-3p, hsa-miR-29b-3p, hsa-miR-32-5p, hsa-miR-16-5p (4-30 fold) and highly upregulated ones included hsa-miR-221-3p, hsa-miR-409-3p, hsa-miR-193a-5p, hsa-miR-125a-5p (80-330 fold). We focused on miRNA-101-3p as it showed the highest level of downregulation (30 fold) and has been shown to function as an important tumor suppressor in other malignancies. Real time RT-PCR confirmed downregulation of miRNA-101-3p. Moreover, microRNA Data Integration Portal (mirDIP) identified miRNA-101-3p as a putative target for survivin and Luciferase activity assays confirmed binding of miRNA-101-3p to 3'UTR of survivin. In addition, overexpression of miRNA-101-3p downregulated survivin and sensitized MM cells to BTZ-induced apoptosis. Furthermore, silencing of survivin upregulated miRNA-101-3p and increased BTZ-induced apoptosis in MM cell lines both in the absence of BMSCs (Apoptosis range in BTZ-treated conditions: 57.65% ± 4.91 and 28.66% ± 0.78 for si-survivin and scrambled control, respectively, p<0.05) and in the presence of BMSCs (41.23% ± 1.43 and 14.8% ± 0.66, for si-survivin and scrambled control, respectively, p<0.05).CONCLUSIONOur results indicate that BMSCs downregulated miRNA-101-3p and upregulated survivin in MM cells compared to MM cells in the absence of stroma. Silencing of survivin or overexpression of miRNA-101-3p sensitized MM cells to BTZ in the presence of BMSCs. These findings suggest that miRNA-101-3p mediates BTZ response of MM cells in the presence of BMSCs by targeting survivin and disclose a role of survivin-miRNA-101-3p axis in regulation of BMSCs-induced BTZ resistance in MM cells, thus provide a rationale to further investigate the anti-myeloma activity of miRNA-101-3p in combination with BTZ as a potential novel therapeutic strategy in MM. DisclosuresNo relevant conflicts of interest to declare.

Drug Design for Overcoming High-Risk Myeloma and Identification of Novel Binding Proteins to Immune-Modulatory Drugs

PURPOSE: Recent progress of the treatment of multiple myeloma (MM) has significantly improved prognosis. However, the MM patients with high-risk cytogenetic abnormalities still showed significantly shorter survival. The purpose of this study is to find compounds effective for high-risk myeloma with lower toxicity by developing chemical structure of the phthalimide. We also tried to isolate their binding molecules to understand molecular pharmacology of the compounds.RESULTS: (1) Screening and optimization of phthalimide derivatives: We screened library of synthetic phthalimide derivatives by their ability to induce apoptosis of MM cell line, KMS34, which has high-risk cytogenetic abnormalities such as t(4;14) and TP53 gene deletion. We finally found TC11, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione, which showed the most potent tumor growth inhibition. Based on the results of screening of phthalimide library, we found the two important points of chemical structure: (i) A 4-amino group lacked growth inhibitory effect, and thus, 5-amino branch is necessary for strong anti-tumor activity. (ii) Modification of 2,6-diisopropylphenyl in phenyl ring of TC11 significantly decreased inhibitory effects of tumor cell growth.(2) Anti-myeloma effects of phthalimide derivatives: TC11 showed significant growth inhibition of all myeloma cell lines (IC50=3-8μM) including those with high-risk cytogenetic changes. Lenalidomide also significantly inhibited growth of myeloma cell lines. However, 30μM of lenalidomide failed to inhibit growth of KMS34 and KMS28 cell lines that have high-risk cytogenetic alterations. TC11 also inhibited growth of bone marrow myeloma cells obtained from the MM patients.(3) Anti-myeloma effects of TC11 in vivo: Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous plasmacytoma in human myeloma cell (KMS34 and KMS11)-bearing SCID xenografts. In the pharmacokinetic analyses, the Cmax was 2.1μM at 1 h after the injection of TC11, with 1.2 h as the half-life. Cmax was 18.1μM at 1.5hr (Tmax), and T1/2 was 4.5hr, when 100mg/kg of TC11 was injected. Cmax was 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr, if 20mg/kg was injected. In oral administration of 200mg/kg of TC11 in lcr mice, Cmax was 3.81μM at 8.0hr (Tmax), and T1/2 was 2.77hr.(4) Toxicity of TC11 and lenalidomide: When 20mg/kg of TC11 was injected to SCID-mice for two weeks, weight loss compared with control mice was not observed. Clonological assay using mice bone marrow cells showed that 10μM TC11 did not suppress the colony formation while 5μM lenalidomide significantly decreased the colony out put. Therefore, TC11 in the concentrations that induced apoptosis of myeloma cells unlikely caused systemic and hematological toxicity.(5) Binding molecules: To understand the molecular mechanism, we employed our unique in vitro screening system using mRNA display, in vitro virus (IVV) method. It was found that TC11 directly bound to nucleophosmin 1 (NPM1) and α-tubulin. Knock-down of NPM1 gene in myeloma cells significantly delayed tumor cell growth. However, cereblon (CRBN), which was considered to be associated with teratogenicity of thalidomide, was not included in TC11-binding molecules in IVV assay. TC11 lacked glutarimide moiety to which CRBN was reported to bind. Thus, TC11 revealed anti-myeloma activity in a CRBN-independent pathway and is conceptionally expected as a non-teratogenic thalidomide-related compound. Interestingly, lenalidomide also directly bound to NPM1. Further analyses were needed to elucidate biological significance of interaction of IMiDs with NPM1 and α-tubulin.CONCLUSION: Our results suggested the possibility that phthalimide derivatives induce tumor cell death independent of CRBN pathway. It was also suggested that drug design and modification of chemical structure of phthalimide enable us to develop further new thalidomide derivatives which have more potent antitumor activity and less toxicity. DisclosuresIchikawa:Takeda Pharmaceutical Company: Research Funding. Matsushita:Takeda Pharmaceutical Company: Research Funding. Hattori:MSD company: Research Funding; Ono Pharmaceutical company: Research Funding; Takeda Pharmaceutical Company: Consultancy; Novo Nordisk company: Research Funding; Mitsubisi Tanabe Pharm: Research Funding; Cosmic Corporation: Research Funding.

Antagonizing HDAC6 Activity: A New Strategy to Maximize the Anti-Myeloma Effects of BET Inhibition

Multiple myeloma (MM) is an incurable plasma cell malignancy and represents the second most common adult hematologic malignancy in the United States. MM is relatively asymptomatic during its early stages and as a result, the majority of patients have advanced disease at diagnosis. Innovations in the treatment of MM, including the development of proteasome inhibitors such as bortezomib (Velcade) have improved clinical outcomes. However, many patients fail to respond to these agents or relapse after initial response highlighting the need for novel therapeutic strategies. Constitutive activation of the MYC oncogene is a frequent pathogenic event in MM that drives disease progression. Aberrant MYC transcriptional activity can increase the levels of a number of factors that are associated with disease progression and drug resistance making it an appealing therapeutic target. Recent studies have demonstrated that inhibition of bromodomain and extra terminal (BET) protein family members including BRD4 decreases the expression of c-MYC and other key oncogenic factors. Here, we demonstrate that shRNA-mediated knockdown of BRD4 or treatment with the BET antagonist JQ1 decreased the expression of c-MYC, BCL-xL, and BCL-2, induced p21 levels, diminished cell viability, and triggered apoptosis in MM cell lines. Comprehensive gene expression profiling of the pharmacodynamic effects of JQ1 revealed that the histone deacetylase HDAC6 was also highly significantly elevated in all MM cell lines and primary patient specimens treated with this agent. Several earlier studies demonstrated that aberrant HDAC6 expression/activity contributes to malignant progression and resistance to a number of classes of anticancer agents including proteasome inhibitors. Based on the roles of HDAC6 in malignant pathogenesis, we hypothesized that its induction may reduce the anti-myeloma activity of JQ1. To test this hypothesis, we utilized both genetic and pharmacological approaches to impair HDAC6 function [shRNA-mediated knockdown of HDAC6, the pan-HDAC inhibitor vorinostat, and the HDAC6-selective inhibitor ACY-1215 (rocilinostat)] and evaluated the consequential impact on the anti-MM effects of JQ1. Notably, antagonzing HDAC6 activity synergistically enhanced the activity of JQ1 in a panel of MM cell lines. These effects were also observed in primary CD138+ cells obtained from patients with MM in a manner that was not affected by prior treatment history. The increased efficacy of these therapeutic combinations was associated with further reductions in c-MYC, BCL-2, and BCL-xL along with significant increases in apoptosis induction as evidenced by enhanced caspase-3 cleavage and DNA fragmentation. Importantly, administration of ACY-1215 was very well tolerated (less than 5% mean transient reduction in body weight) and significantly augmented the in vivo anti-myeloma activity of JQ1 in the RPMI-8226 MM xenograft model as disease burden in combination treated animals was substantially lower than those that received either monotherapy. Immunohistochemical analyses demonstrated that the combination of JQ1 and ACY-1215 led to significantly lower MM cell proliferation (PCNA), increased apoptosis (active caspase-3), and diminished expression of c-MYC and BCL-2. These data suggest for the first time that induction of HDAC6 may represent a key mechanism that promotes drug resistance and limits the efficacy of bromodomain inhibitor therapy. Taken together, our findings demonstrate that abrogation of HDAC6 activity with ACY-1215 or vorinostat is a novel approach to augment the efficacy of bromodomain inhibitors in MM that warrants further investigation. DisclosuresCarew:Boehringer Ingelheim: Research Funding.

Abstract 1017: Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines

Abstract Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, providing a selective advantage for tumor cell survival, due in part, to its ability to inhibit cell death via APAF1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM). Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis. Methods: The expression of HSP70 and TRIAP1 genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1) - three independent transductions, in triplicate. Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry with annexin V and propidium iodide (PI) staining. We also evaluated APAF1 and Caspase9 genes expression by qPCR and Caspase 9 and Caspase 3/7 protein activity. Results: The cell lines RPMI8226 (without deletion of TP53 by FISH) and U266 (deletion of one allele of TP53 by FISH) were chosen for the transduction experiments because they showed significant levels of expression of HSP70 and TRIAP1. The efficiency of transduction, with GFP reporter gene, was 70% for both cell lines. Silencing of both genes was confirmed by qPCR and Western blotting. Inhibition of TRIAP1 increased the percentage of cells in late apoptosis (p&amp;lt;0.001 for RPMI8226 and P&amp;lt;0.01 for U266) and was accompanied by increased expression of Caspase9 in both MM cell lines (p&amp;lt;0.001 for RPMI8226 and p&amp;lt;0.05 for U266). Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines (p&amp;lt;0.01 for RPMI8226 and p&amp;lt;0.05 for U266) and these results were confirmed by increased activity of Caspase3/7 (p&amp;lt;0.01 for RPMI8226 and p&amp;lt;0.05 for U266). Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact of this method on the induction of late apoptosis, through APAF1/Caspase9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever FISH status for TP53 (Support by FAPESP 2010/17668-6). Citation Format: Veruska Lia Fook Alves, Daniela B. Zanatta, mariana bleker oliveira, angela isabel pereira eugenio, rodrigo carlini fernando, Bryan E. Strauss, Gisele Wally Braga Colleoni. Silencing of apoptosome regulating genes, HSP70 and TRIAP1, induces apoptosis in MM cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2015-1017

Abstract 1730: TAS4464, a novel highly potent NEDD8 activating enzyme inhibitor, demonstrates antitumor activity in multiple myeloma through the inactivation of NF-κB pathways

Abstract Background: NEDD8 activating enzyme E1 (NAE) regulates NEDD8 conjugation in the ubiquitin-proteasome system. NAE activates cullin-RING ligases (CRL) and controls the degradation of their substrates, which play key roles in cell cycle progression, DNA replication and signal transduction via various pathways. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of multiple myeloma (MM). MM is highly treatable but rarely curable, and patients with refractory MM would benefit from additional therapeutic options. NF-κB is activated through two signaling cascades, canonical and non-canonical pathways, which are regulated by the CRL substrates, phosphorylated IκBα (p-IκBα) and phosphorylated p100 (p-p100), respectively. Here, we report on the possible use of an NAE inhibitor that we discovered, TAS4464, to treat MM. Materials and methods: TAS4464 was designed and synthesized at Taiho Pharmaceutical Co., Ltd. Human MM cell lines, KMS-11, KMS-12-BM, KMS-26, KMS-34, and MM.1S were used to evaluate the biological activities of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. Effects on the cell cycle were analyzed by using fluorescence-activated cell sorting. The effects of TAS4464 on NEDD8 conjugation and the levels of CRL substrates were evaluated by means of Western blotting. The transcription levels of NF-κB-targeted genes were assessed by using qRT-PCR. Activated p65 and RelB (subunits of NF-κB) were quantified by using commercial DNA-binding ELISA kits. The antitumor activity of TAS4464 was evaluated in an MM.1S xenograft model. Results: TAS4464 inhibited cullin neddylation, leading to cell-growth arrest and apoptosis in MM cell lines in the nanomolar range. These effects were not attenuated after stimulation with interleukin 6 or insulin-like growth factor 1 or in co-culture with stroma cells. TAS4464 led to the accumulation of p-IκBα and p-p100 with subsequent suppression both p65 and RelB activity, whereas bortezomib suppressed only RelB activity. Once-weekly intravenous administration of TAS4464 showed much stronger antitumor activity than that of bortezomib in an MM.1S xenograft model, with inhibited cullin neddylation and induced the cleaved forms of caspases 3 and 8 in the tumors.Conclusion: TAS4464 demonstrated marked antitumor activity in a human MM xenograft model. In addition, TAS4464 inhibited canonical as well as non-canonical pathways of NF-κB, whereas bortezomib inhibited only non-canonical pathway of NF-κB. Therefore, TAS4464 may be a valuable addition to current options for the chemotherapy of MM. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Shingo Tsuji, Takamasa Suzuki, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Yoshikazu Iwasawa, Teruhiro Utsugi. TAS4464, a novel highly potent NEDD8 activating enzyme inhibitor, demonstrates antitumor activity in multiple myeloma through the inactivation of NF-κB pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2015-1730

Anti-β₂-microglobulin monoclonal antibodies overcome bortezomib resistance in multiple myeloma by inhibiting autophagy.

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) have strong and direct apoptotic effects on multiple myeloma (MM) cells, suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the anti-MM effects of combination treatment with anti-β2M mAbs and bortezomib (BTZ). Our results showed that anti-β2M mAbs enhanced BTZ-induced apoptosis of MM cell lines and primary MM cells. Combination treatment could also induce apoptosis of BTZ-resistant MM cells, and the enhanced effect depended on the surface expression of β2M on MM cells. BTZ up-regulated the expression of autophagy proteins, whereas combination with anti-β2M mAbs inhibited autophagy. Sequence analysis of the promoter region of beclin 1 identified 3 putative NF-κB-binding sites from -615 to -789 bp. BTZ treatment increased, whereas combination with anti-β2M mAbs reduced, NF-κB transcription activities in MM cells, and combination treatment inhibited NF-κB p65 binding to the beclin 1 promoter. Furthermore, anti-β2M mAbs and BTZ combination treatment had anti-MM activities in an established MM mouse model. Thus, our studies provide new insight and support for the clinical development of an anti-β2M mAb and BTZ combination treatment to overcome BTZ drug resistance and improve MM patient survival.

Canonical Wnt pathway inhibitor ICG-001 induces cytotoxicity of multiple myeloma cells in Wnt-independent manner.

Canonical Wnt signaling has been implicated in the regulation of multiple myeloma (MM) growth. Here, we investigated whether the targeting of this pathway with a novel pharmacological inhibitor ICG-001 would result in an anti-tumor effect and improvement of chemosensitivity in MM. As expected, ICG-001 specifically down-regulated β-catenin/TCF-mediated transcription in MM cells. Treatment with ICG-001 resulted in growth arrest and apoptosis in MM cell lines and primary MM cells. Moreover, ICG-001 enhanced the cytotoxic effects of doxorubicin and melphalan and abrogated chemoresistance of MM cells to these chemotherapeutics induced by bone marrow stroma. The cytotoxic effect of ICG-001 was caspase-dependent and mediated through transcriptional up-regulation of BH3-only pro-apoptotic members of the Bcl-2 family Noxa and Puma but not through inhibition of canonical Wnt signaling. ICG-001 selectively induced apoptosis in primary MM cells but did not affect non-MM cells of the bone marrow microenvironment. Experiments using a xenograft model of MM showed substantial anti-tumor effects of this compound in vivo. Thus, our study demonstrated that the small molecule inhibitor ICG-001 has strong anti-MM effects and could be developed further for therapeutic intervention in this disease.

Proline-Rich Tyrosine Kinase (Pyk2) Promotes Tumor Progression in Multiple Myeloma (MM) and Represents a Novel Target for Therapy in MM

Background. Proline-rich tyrosine kinase (Pyk2) is a non-receptor tyrosine kinase which belongs to the focal adhesion kinase (FAK) family. It is known to facilitate the TNFα induced EMT process in solid tumors, but this has not been investigated in the field of hematologic malignancies. We therefore dissected the role of PyK2 in multiple myeloma (MM) by looking at its ability to modulate MM biology both in vitro and in vivo.Methods. Lentiviral packaged small hairpin RNA (shRNA), overexpression plasmid, related scrambled shRNA probe and empty vector were introduced into the MM1.S (GFP+/Luc+) cell line, to generate stable Pyk2 knock down (K.D.; #A2 and #A4), Pyk2 over-expression (Pyk2+), and control cells, respectively. The efficiency of K.D. or over-expression was validated by qPCR and immunoblotting. Cell viability and cell proliferation were detected by using CellTiter-Glo® luminescent assay and thymidine uptake, respectively. Gain- and loss-of function studies were also performed on MM cells in the presence of primary bone marrow stromal cells isolated from MM patients (MM-BMSCs). Adhesion of Pyk2 stable cells to fibronectin was measured by using an ECM cell adhesion assay kit. The synergistic effects of Pyk2 with Bortezomib were determined through calculating the DNA synthesis of Pyk2 K.D. cells treated with Bortezomib (2.5-5 µM), using Calcusyn software and Chou-Talalay method. Pyk2 K.D. stable cells were intravenously injected into SCID-Biege mice to generate a xenograft model. In vivo tumor growth was monitored by Bioluminescent Imaging. Pyk2-dependent-modulation of the Wnt/β-catenin pathway signaling was assessed using immunoblotting. The FAK/Pyk2 kinase inhibitor, VS-4718, was tested in vitro using both MM cell lines and primary bone marrow-derived MM cells; and in vivo using bioluminescence imaging.Results. Knockdown of Pyk2 in MM cells significantly repressed cell viability and proliferation, as well as their adhesive ability to BMSCs, compared to scrambled shRNA control cells. Moreover, Pyk2 knockdown induced de-adhesion of MM cells from BMSCs thus inducing chemosensitivity of tumor cells to Bortezomib. We next corroborated our findings by studying Pyk2+ MM cells, and showed that stably upregulated Pyk2 expression promoted MM cell growth as measured by either ATP quantitation or DNA synthesis. Upregulation of Pyk2 expression also stabilized the adhesion of MM cells to BMSCs, leading to drug resistance of MM cells to Bortezomib, compared with vector control cells. Pyk2-regulated tumor growth was further validated by establishing a xenograft mouse model. By using bioluminescence imaging, we found a significantly lower tumor burden in mice injected with Pyk2 K.D. cells, compared to control mice (injected with scrambled shRNA cells). We next dissected the effect of Pyk2 in modulation of cellular signaling in MM cells by using immunoblotting, and demonstrated that Pyk2 played an important role in regulating β-catenin signaling. Indeed, knockdown of Pyk2 induced GSK3β phosphorylation, leading to increased β-catenin phosphorylation, thus resulting in β-catenin degradation and inhibited translocation to the nucleus. Importantly, Pyk2 K.D. cells presented with reduced expression of c-myc and cyclin D1 at the protein level. Conversely, Pyk2 overexpression enhanced β-catenin expression together with c-myc and cyclin D1 up-regulation, thus confirming the role of Pyk2 in modulating Wnt/β-catenin signaling activity in MM. We finally determined that VS-4718, a potent FAK/Pyk2 kinase inhibitor currently in a phase I clinical study, induced apoptosis in MM cell lines and primary MM cells, and inhibited MM tumor growth in vivo.Conclusion. These findings indicate that Pyk2 exhibits pro-oncogenic properties in MM through modulation of Wnt/β-catenin signaling. The findings that the FAK/Pyk2 kinase inhibitor VS-4718 exerts anti-MM activity in cellular and in vivo models support MM as a potential clinical direction for this agent. DisclosuresRing:Verastem: Employment. Tam:Verastem: Employment. Xu:Verastem: Employment. Pachter:Verastem Inc.: Employment, Equity Ownership. Ghobrial:Onyx: Advisory board Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

MLN4924, an NAE inhibitor, suppresses AKT and mTOR signaling via upregulation of REDD1 in human myeloma cells

AbstractThe function and survival of normal and malignant plasma cells depends on the elaborately regulated ubiquitin proteasome system. Proteasome inhibitors such as bortezomib have proved to be highly effective in the treatment of multiple myeloma (MM), and their effects are related to normal protein homeostasis which is critical for plasma cell survival. Many ubiquitin ligases are regulated by conjugation with NEDD8. Therefore, neddylation may also impact survival and proliferation of malignant plasma cells. Here, we show that MLN4924, a potent NEDD8 activating enzyme (NAE) inhibitor, induced cytotoxicity in MM cell lines, and its antitumor effect is associated with suppression of the AKT and mammalian target of rapamycin (mTOR) signaling pathways through increased expression of REDD1. Combining MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MM cell lines which can overcome the prosurvival effects of growth factors such as interleukin-6 and insulin-like growth factor-1. Altogether, our findings demonstrate an important function for REDD1 in MLN4924-induced cytotoxicity in MM and also provide a promising therapeutic combination strategy for myeloma.

Anti-β2 Microglobulin Monoclonal Antibodies Overcome Bortezomib-Induced Drug Resistance In Multiple Myeloma By Inhibition Of Autophagy

Chemotherapy is the most effective treatment for multiple myeloma (MM). Several new drugs have been developed to prolong MM patient survival. However, application of these drugs, such as bortezomib (BTZ), usually induces drug resistance, and patients are prone to quick relapse. It is known that cell death or proliferation is regulated by the crosstalk between apoptosis and autophagy, and autophagy activation inhibits apoptosis by reducing caspase cleavage. Recent studies have shown that autophagy activation plays a role in chemotherapy drug resistance in cancer patients, in particular, BTZ treatment activates autophagy in MM, indicating that inhibition of autophagy could overcome BTZ-induced drug resistance. We have recently reported the generation of monoclonal antibodies (mAbs) against human beta2 microglobuline (b2M), and have found that high dose mAbs have strong apoptotic effects on MM. In this study, using a BTZ treatment-setting, we hypothesized that anti-β2M mAbs could overcome drug resistance and enhance chemotherapy efficacy in MM by inhibiting autophagy. To investigate the combined effects of mAbs and BTZ, MM cells were cultured with addition of BTZ (5 nM) or mAbs (clone D1; 10 mg/ml) or both for 24 hours. Results from Annexin-V binding assay indicated that combined treatment significantly enhanced apoptosis in MM cell lines, such as ARK, ARP-1, MM.1S, and U266, and in primary MM cells as compared with individual treatment, respectively. Next, BTZ-sensitive or -resistant MM cells KAS-6 WT/V10R or OPM-2 WT/V10R were used to determine the importance of BTZ in the synergistic effects, and such synergistic effects was only shown in apoptosis of BTZ-sensitive cells, but not in BTZ-resistant cells. Furthermore, combined treatment significantly increased apoptosis in β2M-overexpressing, but decreased in β2M-knocking down MM cells, which indicated that the synergistic effects is dependent on the surface β2M expression on MM cells. Mechanistic studies further showed that BTZ treatment resulted in an accumulation of cleaved-caspase 9/3 and PARP cleavage, suggesting that combined treatment enhanced caspase activation. In addition, Western blot analysis showed that BTZ treatment upregulated the expression of autophagy proteins, such as LAMP-1, Beclin 1, and LC3B, in a dose dependent manner, whereas combined treatment decreased their expression. Beclin 1, which is a key protein required for autophagy, has been reported to have a potential NF-kB p65 binding site on its promoter. Therefore, we checked if mAbs inhibited BTZ-induced autophagy via NF-kB signaling pathway. Our data showed that BTZ treatment increased p65 nuclear translocation and the levels of phosphorylated p65 in MM cells, while combined treatment significantly reduced both. ChIP assay has further verified that mAb treatment inhibited p65 binding to Beclin 1 promoter. To examine our hypothesis in vivo, MM cells ARP-1 or MM.1S were subcutaneously injected into SCID mice. The mice were then subcutaneously injected with mAbs (0.6 mg/kg) or intraperitoneally injected with BTZ (0.1 mg/kg) individually or in combination. Tumor burdens were assessed by measuring tumor volumes and serum M-protein levels by ELISA. The combination of anti-β2M mAb and BTZ treatment repressed tumor growth and prolonged the survival of tumor-bearing mice as compared with individual treatment. In conclusion, our study for the first time demonstrated that anti-β2M mAbs prevent BTZ resistance and enhance its anti-MM efficacy by reducing the expression of autophagy proteins via NF-kB signaling. Thus, our studies provide a new insight into clinical development of anti-β2M mAbs to overcome chemotherapy drug resistance and improve MM patient survival. Disclosures:No relevant conflicts of interest to declare.

Combined Carfilzomib and Selective PI3Kδ Inhibition (TGR1202) Results In Enhanced Myeloma Cell Apoptosis

IntroductionThe PI3K signaling pathway plays a vital role in regulating cell growth, proliferation and survival in multiple myeloma (MM) as well as in many other cancers. TGR-1202, an isoform-specific PI3Kδ inhibitor, with efficacy in preclinical models of hematologic malignancies, is currently in Phase I clinical development. In multiple myeloma, PI3K signaling appears to be very important for many extracellular signals, yet inhibition with -pan PI3K inhibitors have not exhibited significant activity. However, literature reports indicate that there are several MM cell lines that express PI3K -δ, and do appear to have differential sensitivity to specific isoform inhibition as opposed to pan PI3K inhibition. In this report, we sought to evaluate the effects of TGR-1202 alone and in combination with the proteasome inhibitor carfilzomib, with the intent of further understanding the mechanism of action and evaluating the impact of the combination. MethodsHuman myeloma cell lines (MM.1S, MY5, RPMI8226, U266, KMS11, ARH-77, OPM1, OPM2, LP1, JJN3 and L363) were treated with TGR-1202 alone, carfilzomib alone, or with the combination of TGR-1202 and carfilzomib. Annexin V/PI staining and Western blot were used to identify the cellular and molecular sequelae of the combination. Result10 µM TGR-1202 alone did not cause significant cell death in the MM cell lines tested at 48 hours. When cells were treated with the combination of TGR-1202 and 3 nM carfilzomib, we observed enhanced apoptosis in all of the tested cell lines. In the U266 cell line, 3 nM carfilzomib and 10 µM TGR-1202 induced 16% and 14% cell apoptosis respectively. In the combination treatment, apoptosis increased to 75%. To explore the molecular mechanisms underlying the combination, we used a Western blot assay to evaluate the impact of the combination on the mTOR signaling pathway, a known reciprocal feedback loop when PI3K is blocked. TGR-1202 alone did not have an obvious effect on the mTOR signaling pathway, yet combining TGR-1202 with carfilzomib, significantly inhibited phospho-mTOR, suggesting total pathway blockade. ConclusionThe combination of TGR-1202 with carfilzomib induces synergistic apoptosis in MM cell lines. Preliminary data suggests that this occurs through blockade of the entire reciprocal feedback loop of mTOR activation. Additional data from primary patient samples and underlying mechanisms will be presented at the meeting. These findings support the rationale for future clinical studies of TGR-1202, a selective PI3K-δ inhibitor in combination with the proteasome inhibitor carfilzomib. Disclosures:Boise:Onyx: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.

Overcoming CD28-Mediated Multi-Drug Resistance With a Novel PIM2 Inhibitor

Drug resistance in multiple myeloma (MM) is the major cause of treatment failure and is significantly mediated by pro-survival interactions with bone marrow microenvironment. A key myeloma receptor involved in this interaction is CD28, which has been largely characterized as the prototypic T cell costimulatory receptor. However, CD28 expression on myeloma cells is significantly correlated with disease progression, worse prognosis, and is significantly higher in the poor prognosis t(14;16) MAF subgroup. We now report that CD28 signaling mediates significant drug resistance and protects MM against death from multiple chemotherapeutics with different mechanisms of action – including dexamethasone, arsenic trioxide, melphalan or bortezomib. Inhibition of specific signaling (PI3K or Akt) or targets (Foxo3A or Bim) downstream of CD28 activation abrogates this protection. Unexpectedly, we found evidence that the PIM2 kinase (which is largely uncharacterized in MM but is also significantly overexpressed in the MAF subgroup) may be a previously unreported component of the CD28 pro-MM survival pathway. The novel small molecule PIM2 inhibitor JP_11646 (IC50 0.5 nM) abrogates CD28-mediated protection against apoptosis in MM cell lines in vitro, which we have not previously seen for any chemotherapeutic tested. In addition, blockade of CD28 activation sensitized MM cells significantly to JP_11646-induced death. Altogether, these data suggested that PIM2 inhibitors can overcome a major mechanism of multi drug resistance in MM.Jasco's novel and selective pan-PIM inhibitor (JP_11646) has demonstrated biochemical IC50s of 24, 0.5 and 1 nM for PIM1, PIM2 and PIM3 respectively. The PIM mechanism of action has been confirmed through cell based transphosphorylation assays, where JP_11646 decreased PIM dependent phoshphorylation of the proapoptotic protein BAD at nM levels. JP_11646 increases apoptosis and decreases cell viability in multiple myeloma cell lines with the MAF translocations (<100 nM). JP_11646 is orally bioavailable and has demonstrated in vivo efficacy, inhibiting tumor growth by >80% in a MM1.S tumor xenograft study. These data provide solid rationale for further development of JP_11646 as a targeted therapy in MM, and specifically for patients exhibiting the MAF translocation. Disclosures:Baldino:Jasco pharmaceuticals: Employment, Equity Ownership, Founder and President Other, Membership on an entity's Board of Directors or advisory committees. Caserta:Jasco Pharmaceuticals: Co-Founder Other, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Dumas:Jasco Pharmaceuticals: Employment. Flanders:Jasco Pharmaceuticals: Employment. Lee:Jasco Pharmaceuticals: Research Funding.

Inhibiting Aberrant Signal Transducer and Activator of Transcription Protein Activation with Tetrapodal, Small Molecule Src Homology 2 Domain Binders: Promising Agents against Multiple Myeloma

The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes. In particular, Stat3 protein has attracted attention as it regulates the expression of genes involved in a variety of malignant processes, including proliferation, survival, migration, and drug resistance. Multiple myeloma (MM) is an incurable hematologic malignancy that often exhibits abnormally high levels of Stat3 activity. Although current treatment strategies can improve the clinical management of MM, it remains uniformly incurable with a dismal median survival time post-treatment of 3-4 years. Thus, novel targeted therapeutics are critically needed to improve MM patient outcomes. We herein report the development of a series of small molecule Stat3 inhibitors with potent anti-MM activity in vitro. These compounds showed high-affinity binding to Stat3's SH2 domain, inhibited intracellular Stat3 phosphorylation, and induced apoptosis in MM cell lines at low micromolar concentrations.

Bortezomid enhances the efficacy of lidamycin against human multiple myeloma cells

The proteasome inhibitor bortezomib has been applied successfully to treat multiple myeloma (MM). Its synergistic effects with other anticancer drugs have been studied widely. In the present study, it was found that lidamycin (LDM), a member of the enediyne antibiotic family, showed much more potent cytotoxicity than bortezomib to MM cell lines: U266 and SKO-007. Here, we investigated the potential synergy of bortezomib and LDM on MM cells. The results showed that cotreatment of bortezomib and LDM synergistically induced cytotoxicity and apoptosis in MM cell lines, followed by enhanced caspase-3 cleavage and degradation of poly-ADP-ribose polymerase together with the decreased nuclear factor-κB protein. These two drugs synergistically induced apoptosis, which was associated with enhanced activation of two mitogen-activated protein kinases: p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase. Moreover, bortezomib plus LDM synergistically induced apoptosis was also associated with downregulation of extracellular signal-regulated kinase, and induction of endoplasmic reticulum stress response. Overall, our results indicate that the combined regimen of bortezomib and LDM might be a potential therapeutic remedy for the treatment of MM.

Macrolide Antibiotics Block Autophagy Flux and Sensitize to Bortezomib Via Endoplasmic Reticulum-Stress-Mediated CHOP Induction in Myeloma Cells

Abstract 4992Macroautophagy (hereafter, “autophagy”) is a highly conserved cellular process of self-degradation in eukaryotes. Intracellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intracellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) cell lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes.BZ potently induces autophagy, ER–stress, and apoptosis in MM cell lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins, and induced the proapoptotic transcription factor CHOP. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. BIM, BAX, DR5, and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and up-regulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP−/− MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide.These data suggest that ER-stress mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER-stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy. Disclosures:No relevant conflicts of interest to declare.